Background: Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traﬃc, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the ﬂux through various routes into the early secretory pathway, we quantiﬁed the global and membrane-associated translatomes of Komagataella phaﬃi.
Results: By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantiﬁed demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway.
Conclusion: A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.